EP2123308A1 - Sterilisierung mittels Behandlung mit einem Reduktions- und Oxidationsmittel - Google Patents

Sterilisierung mittels Behandlung mit einem Reduktions- und Oxidationsmittel Download PDF

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EP2123308A1
EP2123308A1 EP08155633A EP08155633A EP2123308A1 EP 2123308 A1 EP2123308 A1 EP 2123308A1 EP 08155633 A EP08155633 A EP 08155633A EP 08155633 A EP08155633 A EP 08155633A EP 2123308 A1 EP2123308 A1 EP 2123308A1
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Prior art keywords
item
enzyme
water
sterilization
reductant
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English (en)
French (fr)
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Jan SØRENSEN
Belinda Dueholm Uckevicius
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BK Medical AS
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BK Medical AS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/20—Gaseous substances, e.g. vapours
    • A61L2/202—Ozone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
    • A61L2/20—Gaseous substances, e.g. vapours
    • A61L2/206—Ethylene oxide

Definitions

  • the present invention relates to the field of sterilization of an item. More particularly, the invention concerns a method, the use of this method, and an apparatus for sterilization.
  • the most used method for sterilisation is autoclaves, where high temperature, often in combination with steam, is used for sterilization. Often, the temperatures in an autoclave exceed 121°C or higher, resulting in damage of temperature sensitive equipment.
  • an item or object is considered sterile, when it is free of all living microorganisms, and this state of sterility is the result of a sterilizing procedure.
  • This may be a chemical and/or physical process destroying and/or eliminating all living organisms, which also includes resistant bacterial spores.
  • the result of a sterilizing process is defined as a probability of less than one in one million that a microorganism has survived on an item. This is also referred to as "sterility assurance level" and is used by the medical device industry to characterize sterilized medical devices. In practical terms and in view of the current invention, sterilization is defined as a 6 log reduction in microbial load.
  • Disinfection is defined as the process of destroying or inhibiting growth of microorganisms.
  • Low-level disinfection kills vegetative, i.e. growing bacteria, fungi and susceptible viruses
  • intermediate-level disinfection kills most bacteria, fungi and viruses, but not bacterial spores.
  • High-level disinfection kills all bacteria, fungi, viruses and may kill bacterial spores, especially when for example prolonged contact times are chosen.
  • Bacterial spores are considered the most resistant of all living organisms because of their ability to withstand a variety of chemical and/or physical processes, which otherwise are capable of effectively destroying all other living organisms. Fungi have also the ability to produce spores, but in contrast to bacterial spores, fungal spores are less resistant.
  • spores are differentiated cells formed within a vegetative bacterial cell in response to unfavourable environmental conditions. It has been reported that spores are several orders of magnitude more resistant to lethal treatments and/or chemical agents than its parent cell.
  • a cross section of a bacterial spore is presented in Fig. 1 .
  • the spore is often surrounded by a covering known as the exosporium (Ex), which overlies the spore coat (SC).
  • the spore coat is a complex, multi-layered structure consisting of more than 50 proteins.
  • the spore coat is the major resistance barrier for a large number of chemicals, such as most oxidizing agents including chlorine dioxide, hypochlorite, ozone and peroxynitrite, but not against heat or radiation.
  • the layered outer coats of a bacterial spore are rather inert and play a predominant role in protecting the spore against exogenous agents. It is known that disulfide bridges are a feature of cellular walls and other protein-containing features of bacterial cells. As much as 80% of the total protein of the spore is made up of keratin-like protein. The stability of keratin structures is due to frequent valence cross links (disulfide bonds) and secondary valence cross links (hydrogen bonds) between neighboring polypeptide chains.
  • Keratin-like proteins are resistant to proteolytic enzymes and hydrolysis, but typically insoluble in aqueous salt solutions or dilute acid or base solutions.
  • the cortex (Cx) which consists of peptidoglycan lies beneath the spore coat separated by the outer membrane (OM).
  • the inner membrane (IM) is located between the core wall and the cortex, and surrounds the core (Co) of the endospore.
  • the inner membrane exhibits an extremely low permeability to small hydrophobic and hydrophilic molecules.
  • the core contains normal cell components, such as DNA and ribosomes, but it is considered metabolically inactive.
  • Bacillus stearothermophilus is a thermophilic species which can grow at temperatures at 65°C or above. Spores of Bacillus stearothermophilus are highly temperature resistant and they are used for example as sterility indicators for steam sterilization. Commercial indicators from FLUKA consist of 1 million spores impregnated on paper strips. These indicators are specified by US military specification MIL-S-36586 and are GMP (Good Manufacturing Practice) requirements of the US FDA (Food and Drug Administration). While Bacillus stearothermophilus is mainly used for testing sterilization at high temperatures, Bacillus atrophaeus is used as indicator for sterilization at low temperature.
  • the Commercial indicators can be obtained from Raven Biological Laboratories, INC and consist of 1 million spores on stainless steel discs. This microorganism is also part of an US FDA-approved method (FDA Guidance on Premarket notification 510K submissions for Sterilizers intended for use in the health care facilities).
  • Sterilization procedures can be divided in two major groups, namely physical and chemical processes.
  • UV light form of non-ionizing radiation
  • UV is used for sterilization at room temperature, but it is limited to surfaces and some transparent objects. UV is mainly used for sterilizing the interiors of biological safety cabinets between uses. UV is ineffective for sterilization in shaded areas, e.g. cavities or areas under dirt. UV damages many plastics.
  • Microwave can also be used for sterilization, where the non-ionizing radiation produces energy rich hyperthermic conditions that disrupt life by acting on water molecules, thereby disrupting e.g. cell membranes. Short sterilization cycles of few minutes at only slightly elevated object temperatures can be achieved. Not all objects are suited for sterilization by microwave.
  • Energy-rich, ionizing radiation in form of ⁇ -particles, X-rays or ⁇ -rays is routinely used mainly for batch sterilization, where the ionic energy of the radiation is converted to thermal and chemical energy. Sterilization cycles are long, often overnight. Major advantage is the ability of the ionizing radiation to penetrate through larger objects. Furthermore, dosimetry can be used for immediate assessment of the efficiency of the sterilization process, instead of depending on tedious microbiological tests.
  • Chemical sterilization procedures are usually applied when the items to be sterilized are heat or moisture sensitive, i.e. when they cannot be sterilized in a dry or steam autoclave.
  • Common chemical agents that are used for killing microorganisms including bacterial spores are ethylene oxide gas, formaldehyde gas, glutaraldehyde activated solution or gas, hydrogen peroxide plasma/vapor, peracetic solution, bleach and ozone gas or in aqueous solution.
  • the major disadvantages of chemical sterilization are (i) the toxicity and (ii) flammability/explosive danger of the chemical compounds used, (iii) the need for aeration of sterilized items after sterilizitation (iv) their often aggressive and corrosive properties towards e.g. plastics and metals, and finally, (v) the long sterilization times needed.
  • PCT/DK/2007/000486 a patent application submitted by the same applicant, concerns a method and apparatus for two-step sterilization, comprising the steps of contacting an item or part of an item with a water-based fluid containing at least one enzyme, and a substantially water-free environment with a gas having oxidative properties, such as ozone.
  • the applicant has discovered a process for sterilization and/or disinfection suitable for killing bacterial spores, said process comprising the steps of (i) treatment with a water-base fluid comprising a reductant, followed by a (ii) treatment with a fluid comprising an oxidant.
  • the invention solves the problem of sterilizing and/or disinfecting items which are prone to be impaired or damaged by for example one or more of temperature, pH, positive or negative pressure, radiation, oxidation, detergent and/or enzymatic activity.
  • a first aspect of the invention concerns a method for sterilization, comprising the steps of contacting an item or part of an item with a water-based fluid containing a reductant, followed by contacting said item or part of an item with a gas having oxidative properties in a substantially water-free environment, thereby providing sterilization and/or disinfection of said item or part of said item.
  • a second aspect of the invention concerns the use of said method, such as treatment of one or more items, or one or more parts of an item selected from the group consisting of laboratory item, medical item, dental item, military item, biological item, food processing related item, satellite and space rocket.
  • a third aspect of the invention relates to an apparatus comprising the necessary means for performing, using or applying said method.
  • the present invention which completely or partially satisfies the abovementioned objectives, relates to a method comprising two distinct steps/treatments for sterilization and/or disinfection.
  • the first step (a) renders a layer or fraction or subfraction of a layer or coat of the microbe or bacterial spore susceptible for the sterilizing/disinfecting action of the second treatment (b).
  • the inventors surprisingly and unexpectedly discovered that sterilization and/or disinfection was achieved by treatments with an aqueous fluid under reducing conditions, such as water comprising a reductant, followed by treatment under oxidizing conditions, such as a treatment with a gaseous fluid comprising an oxidant, such as air comprising ozone. This was feasible at temperatures well below the boiling point of water. Furthermore, sterilization can be achieved in a surprisingly short time. Finally, this method proves suitable for sterilization of items or part of items that are not suitable for conventional sterilization/disinfection procedures.
  • the invention relates to a method according to claim 1, providing a method for sterilization or disinfection, comprising the steps of contacting one or more item or part of said item with (a) a water-based fluid comprising at least one reductant, and (b) a substantially water-free environment with a gas having oxidative properties, wherein step (a) precedes step (b).
  • the method comprises a further step (a*) of contacting the one or more item or part of said item to be sterilized with a water-based fluid comprising at least one enzyme, wherein said step (a*) precedes step (b).
  • step (a*) precedes step (a).
  • step (a) precedes step (a*).
  • the method suitable for sterilization and/or disinfection comprising the steps of contacting one or more item or part of an item with a water-based fluid containing a reductant, and in another distinct step which is performed after the previous step, contacting the one or more item or part of an item with an oxidant, such as a gas with oxidative properties in a substantially water-free environment.
  • an oxidant such as a gas with oxidative properties in a substantially water-free environment.
  • a water-base fluid according to the invention comprises water as the main component, such as at least 80%, 90%, 95%, 99%, or 99.5% (volume/volume) water.
  • the water-based fluid comprise one or more of component, chemical composition, enzyme, additive and the like in any form, such as e.g. as solution, suspension, dispersion, and/or emulsion, or any combination of solution, suspension, dispersion, and/or emulsion of one or more of any component, composition, enzyme, additive and the like.
  • the water-based fluid can be 100% water, such as distilled water, or clean water, such as filtered and/or de-ionized water. According to one embodiment of the invention, the water based fluid is based on processed water.
  • step (a) and/or (a*) comprise(s) an ultrasonic treatment.
  • treatment with a water-based fluid comprises one or more of ultrasonic treatment, sonication, mixing, vortexing, moving, agitating and/or pumping an aqueous fluid.
  • the ultrasonic treatment/sonication, and/or mixing, vortexing, moving and/or pumping liquid solubilises dissolves, and/or distributes compounds or particles within said water-based fluid.
  • These compounds and/or particles including spores, microorganisms, biological material and/or dirt
  • said ultrasonic treatment is facilitating this process.
  • one or more rinsing step(s) is included before step (a), step (a*), and/or step (b).
  • a rinsing and/or washing step can comprise rinsing with processed water and/or processed gas.
  • one or more rinsing steps can be provided between different steps of treatment, such as between treatment with a water based fluid comprising a reductant, and treatment with a gas with oxidative properties.
  • Such one or more rinsing steps may consist of rinsing, flushing or treatment with processed water and/or processed gas, and/or other methods known in the art.
  • the purpose of such a rinsing step can be removal of one or more undesired compounds, including dirt, biological material, contamination, chemical composition, buffer, buffering agent, acid, base, salt, OH - , H + , reductant, oxidant, water, enzyme, enzyme inhibitor, detergent, and the like, and any combination thereof.
  • the biological material is one or more of (biological) tissue, biomass, living or dead biological matter, biomaterial, bio-compatible material, bio-applicable material; biomolecule (i.e. a chemical compound or composition that naturally occurs a living organism), biotic material, processed biotic material, cellular component, material/substances of which a cell is composed, organic material/matter, viable material (i.e. material capable of living, developing, and/or germinating under appropriate condition), and/or body fluid.
  • Processed water according to the present invention can be e.g. demineralised water, tap water or sterile water. Processed water does not need to be sterile, if the microorganisms present therein are killed by the subsequent ozone treatment.
  • the processed water is sterile-filtered, such as passing said water through a filter with a defined maximal pore size. A filter with pore size 0.2 ⁇ m can effectively remove bacteria. If viruses must also be removed, a much smaller pore size may be needed, such as around 20 nm or less.
  • the processed water is sterilized by heating, such as autoclaving, or by a step of evaporation followed by condensation.
  • the processed water can be sterilized by a combination of any treatments known in the art, or combination of treatments known in the art, or any of the aforementioned steps or processes.
  • processed water can comprise one or more additives.
  • the aqueous fluids in step (a) and (a*) are based on processed water, where e.g. one or more of reductant(s), enzyme(s), additive(s), and the like, and any combination thereof are added.
  • the method comprises one or more rinsing step(s) which comprises contacting said one or more item or part of said item with processed water and/or processed gas.
  • a substantially water-free environment can be defined as an environment with a low level of humidity, such as the lumen and/or inner surfaces of a chamber. This can also refer to a reaction chamber or container, its lumen and/or its content, which does not contain visible traces of humidity (such as droplets or pools of water). Alternatively, a substantially water-free environment refers to the content of a container or reaction chamber after removal of water-based fluid. Low level of humidity means less than 60%, 50%, 40%, 33%, 25%, 20%, 15%, 10%, 5%, 2%, 1%, 0.1% or 0.01% relative humidity.
  • low level of humidity refers to ambient humidity, such as around 40-60% relative humidity.
  • a processed gas can be sterile, sterile-filtered, and/or sterilized (e.g. by UV).
  • Processed gas can for example be dry or dried air, ambient air or nitrogen, and this gas could also comprise ozone.
  • the processed gas is ambient air with a low level of humidity, i.e. a lower level of humidity than ambient air.
  • the processed gas does not contain any spores or other forms of life that are not killed by the succeeding ozone treatment.
  • a processed gas comprising or consisting of dry or dried air can provide a substantially water-free environment according to the invention.
  • the processed gas is sterile-filtered, such as passing said gas through a filter with a defined maximal pore size.
  • a filter with pore size 0.2 ⁇ m is believed to effectively remove bacteria. If viruses must also be removed, it can be appropriate to use a smaller pore size, such as around 20 nm or less.
  • the processed gas can be sterilized by a combination of any treatments known in the art, or combination of treatments known in the art, or any of the aforementioned steps or processes disclosed in this specification.
  • reductant can be used interchangeably with the term “reducing agent” and/ or “reducer”, and is meant to comprise a chemical composition with a negative redox potential compared to a standard hydrogen electrode, also called normal hydrogen electrode.
  • reducing agent and/ or “reducer”
  • reductant is oxidized, while the partner in said redox reaction is reduced.
  • a reductant is a chemical composition capable of reducing a disulfide bridge, such as a disulfide bridge between two cysteins.
  • a disulfide bridge can be an intra-molecular disulfide bridge, i.e. a disulfide bridge within a chemical composition, such as a protein molecule.
  • a disulfide bridge can be formed between two different molecules, e.g. proteins, also called inter-molecular disulfide bridge. Said two different molecules can be of identical, similar or different.
  • a molecule can have one or more, inter- and/or intramolecular disulfide bridges. Commonly, disulfide bridges can be formed between two sulphur containing amino acids.
  • Cystein is an amino acid comprising a thiol side chain. When incorporated in a protein, the cystein is capable of forming a disulfide bridge to a second cystein. Because of the high reactivity of this thiol, cysteine is believed to be an important structural and functional component of many proteins and enzymes. A cystein-dimer, linked via a disulfide bridge is also called cystine.
  • the reductant capable of reducing a disulfide bridge comprises one or more chemical composition known in the art for being capable of breaking and/or modifying an intra- or inter-molecular disulfide bridge, e.g. between to cysteins.
  • the reductant is selected from the group consisting of on or more of ⁇ -mercaptoethanol, dithiothreitol (DTT), ascorbic acid, quinone, polyphenol with up to hundreds of polymeric subunits including but are not limited to phenol-rich polymers of flavonoids, gallic acid, ellagic acids and their respective carbohydrate esters, salts and derivatives, proanthocyanidin, including their free acid forms, derivative DTT, derivative of ⁇ -mercaptoethanol, and any combination thereof.
  • DTT dithiothreitol
  • ascorbic acid quinone
  • polyphenol with up to hundreds of polymeric subunits including but are not limited to phenol-rich polymers of flavonoids, gallic acid, ellagic acids and their respective carbohydrate esters, salts and derivatives, proanthocyanidin, including their free acid forms, derivative DTT, derivative of ⁇ -mercaptoethanol, and any combination thereof.
  • the reductant is one or more of dithiothreitol, a derivate of dithiothreitol, ⁇ -mercaptoethanol, a derivate of ⁇ -mercaptoethanol, and any combination thereof.
  • the chemical composition known to be capable of modifying a thiol group is present in a concentration (weight/volume) of about 0.001 % to 5%, or 0.001 % to 1 %, or in a concentration of about 0.005% to 0.5%.
  • the aqueous fluid in step (a) comprises a reductant in a concentration (weight/volume) of less than 0.001 %; 0.001 % to 10%; 0.001 % to 0.005%; 0.005% to 0.01%; 0.01 % to 0.05%; 0.05% to 0.1%; 0.1 % to 0.5%; 0.5% to 1 %; 1 % to 2%; 2% to 5%; or 5 to 10%; or more than 10%.
  • the total concentration of reductants in step (a) is less than 0.001 %; 0.001 % to 10%; 0.001 % to 0.005%; 0.005% to 0.01 %; 0.01 % to 0.05%; 0.05% to 0.1 %; 0.1 % to 0.5%; 0.5% to 1%; 1% to 2%; 2% to 5%; or 5 to 10%; or more than 10%.
  • a reductant is present in step (a) in a molar concentration of 1 mM or less; 1-2 mM; 2-5 mM; 5-10 mM; 10-50 mM; 50-100 mM; 0.1-0.5 M; 0.5 to 1 M; or more than 1 M.
  • the total molar concentration of reductants present in step (a) is 1 mM or less; 1-2 mM; 2-5 mM; 5-10 mM; 10-50 mM; 50-100 mM; 0.1-0.5 M; 0.5 to 1M; or more than 1 M.
  • the resulting thiol group upon reducing of the disulfide bridge by a reductant, is modified in order to prevent (re-)creation of a new disulfide bridge.
  • a modification of a thiol group can comprise the use of one or more chemical composition(s) known in the art, which is/are known to be capable of modifying a thiol group, such as iodoacetamide and iodoacetate.
  • the chemical composition known to be capable of modifying a thiol group is a derivate of iodoacetamide and/or iodoacetate.
  • an alkaline pH is provided and/or maintained during treatment with an aqueous fluid comprising one or more reductant(s), and/or one or more enzyme(s), such as a pH greater than 7.1; 7.5; 8.0; 9.0; 10.0; 11.0; 12.0; 13.0 and/or 14.0.
  • an acid pH is provided and/or maintained during reductant and/or enzyme treatment, such as a pH less than 6.9; 6.0; 5.0; 4.0; 3.0; 2.0; and/or 1.0.
  • a near neutral pH is maintained and/or provided during reductant and/or enzyme treatment, such as a pH range of 6.0-8.0; 6.5-7.5, and/or 6.8-7.2.
  • the pH of a water-based fluid can be in the range of pH 2.0-12.0; 4.0-10.0, 6.0-8.0, or 6.8-7.2.
  • it can be appropriate to maintain a constant pH during one step, and it can be appropriate to change the before or during another treatment step.
  • the pH of the various fluids used during sterilization or disinfection may be similar, or very different.
  • the pH is in the area of the pH optimum of the enzyme, such as +/-1.0, 0.5, or 0.2 around the enzyme's pH optimum, where the pH optimum has been established in the same or similar buffer and/or reaction conditions.
  • a constant pH i.e. a pH +/- 0.05, or a near constant pH (+/- 0.2) is maintained during reductant and/or enzyme treatment.
  • the pH is not maintained constant or near constant.
  • a defined and/or desired pH can be achieved by providing titration with an acid or a base.
  • an appropriate buffer also called buffer solution, can be provided.
  • Such a buffer commonly comprises one or more buffering agent, such as weak acid(s) and/or weak base(s).
  • step (a) is performed at a pH in the range of pH 6.0 to 14.0; 7.0 to 10; 7.0 to 8.0.
  • step (a*) is performed at a pH in the range of pH 4 to 10; 6 to 8; or 6.8 to 7.2.
  • a rinsing step is performed at a pH of in the range of pH 6.0 to 14.0; 7.0 to 10; or 7.0 to 8.0; or pH 6.0 to 14.0; 7.0 to 10; or 7.0 to 8.0.
  • step (a), (a*) and/or a rinsing/washing step is performed at a pH in the range of 1.0 to 2.0; 2.0 to 3.0; 3.0 to 4.0; 4.0 to 5.0; 5.0 to 6.0; 6.0 to 7.0; 7.0 to 8.0; 8.0 to 9.0; 9.0 to 10.0, 10.0 to 11.0; 11.0 to 12.0; 12.0 to 13.0;or 13.0 to 14.0.
  • step (a), (a*) and/or rinsing/washing step are performed at around the same pH or similar pH, or at a different pH.
  • buffering agents and/or buffers are known in the art, and they can comprise chemical compositions like TAPS (3- ⁇ [tris(hydroxymethyl)methyl] amino ⁇ propanesulfonic acid); Bicine (N,N-bis(2-hydroxyethyl)glycine), Tris (tris(hydroxymethyl)methylamine), Tricine (N-tris(hydroxymethyl) methylglycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TES (2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), cacodylate (dimethylarsinic acid), MES (2-(N-morpholino)ethanesulfonic acid), acetate, citrate, carbonate, phosphate, ammonium, a salt of an organic or inorganic
  • oxidant can be used interchangeably with the term “oxidizing agent”, and is meant to comprise a chemical composition with a positive redox potential compared to a standard hydrogen electrode. In a redox reaction, the oxidant is reduced, while the partner in the redox reaction is oxidized.
  • redox is meant to comprise an abreviation for reduction/oxidation.
  • a "redox reaction” is meant to describe a chemical reaction comprising a change in oxidation state and/or oxidation number of one or more chemical compositions, such as molecules, atoms and/or ions involved.
  • a redox reaction can comprise simple redox process such as the oxidation of carbon to yield carbon dioxide, or the reduction of carbon by hydrogen to yield methane (CH 4 ), or the like.
  • CH 4 methane
  • it can be one or more complex process(es), such as the oxidation of a sugar in a cell through a series of potentially complex electron transfer processes.
  • the term “redox” comes from the two concepts of reduction and oxidation.
  • oxidation describes the loss of electrons by a chemical composition, such as molecule, atom and/or ion; while reduction describes the gain of electrons by a chemical composition, molecule, atom and/or ion.
  • oxidation and reduction can also refer to a change in oxidation number, and the actual transfer of electrons may never occur.
  • oxidation can be also defined as an increase in oxidation number, and reduction as a decrease in oxidation number.
  • redox reaction which are classed as "redox" even though no electron transfer occurs, such as those involving covalent bonds.
  • the sterilization process comprises a third step, comprising the use of a water-based fluid containing one or more enzymes.
  • a third step comprising the use of a water-based fluid containing one or more enzymes.
  • addition of such a step comprising at least one enzyme improves disinfection or sterilization, such as increase in log reduction and/or reduction of time necessary for providing disinfection or sterilization.
  • the sterilization process can also comprise a step comprising where the water-based fluid comprises one or more reductant(s) and one or more enzymes.
  • one or more enzymes can be added at any time to e.g. the aqueous fluid comprising one or more reductant.
  • suitable additives can be added in order to provide favourable reaction conditions for the one or more enzyme(s). This can also comprise providing a change in pH.
  • one or more reductant(s) can be added to the aqueous fluid comprising one or more enzymes.
  • suitable additives can be added in order to provide favourable or more favourable reaction conditions for the one or more reductant(s). This can also comprise providing a change in pH.
  • Such an enzyme can be selected from the group comprising microbial cell wall modifying or degrading enzymes, protein modifying or degrading enzymes and/or fat modifying or degrading enzymes. Without limitation, these enzymes can be selected from the group comprising cellulases, chitinases, amylases, proteases, lysozyme(s), phosphatases, kinases and/or lipases. In an embodiment of the invention, the enzyme or enzymes are capable of degrading the cell wall of bacterial spores. More precisely and non exclusively, the enzyme or enzymes are capable of degrading or impairing at least in part the exosporium, the spore coat, the outer membrane, the spore coat and/or the inner membrane.
  • these enzyme or enzymes are capable of impairing the spore and/or the protective layer(s) of the spores to such a degree, that the subsequent treatment with a gas containing ozone results in sterilization and/or disinfection according to the invention.
  • the enzymatic treatment comprises incubation with one or more enzymes selected from the group consisting of cellulose; chitinase; amylase; protease; lysozyme; phosphatase; kinase; lipase; sulfatase (a type of esterase enzyme which removes sulfate from a variety of substrates.
  • one or more enzymes selected from the group consisting of cellulose; chitinase; amylase; protease; lysozyme; phosphatase; kinase; lipase; sulfatase (a type of esterase enzyme which removes sulfate from a variety of substrates.
  • Galactosamine-6 sulfatase N-acetylglucos-amine-6-sulfatase
  • neuraminidase a glycoside hydrolase enzyme (EC 3.2.1.18).
  • aminopeptidase a zinc-dependent enzyme produced by glands of the small intestine and assists in the enzymatic digestion of proteins therein
  • a digestive enzyme produced by a gland of a mammal, such as dipeptidase, maltase, sucrase, lactase, and enterokinase
  • achromopeptidase a lysyl endopeptidase with a MW of ⁇ 27kD.
  • lysostaphin a Staphylococcus simulans metallo-endopeptidase. It can function as an antimicrobial against Staphylococcus aureus.
  • Lysostaphin is a zinc endopeptidase with a molecular weight of approximately 25 kDa.
  • labiase from Streptomyces fulvissimus is an enzyme preparation useful for the lysis of many Gram-positive bacteria such as Lacto-bacillus, Aerococcus and Streptococcus.
  • Labiase contains ⁇ -N-acetyl-D-glucosaminidase and lysozyme activity); mutanolysin provides gentle cell lysis for the isolation of easily degradable biomolecules and RNA from bacteria. It has been used in the formation of spheroplasts for isolation of DNA.
  • Mutanolysin is a 23kD N-Acetyl Muramidase, like lysozyme, is a muralytic enzyme that cleaves the N-acetylmuramyl- ⁇ (1-4)-N-acetylglucosamine linkage of the bacterial cell wall polymer peptidoglycan-polysaccharide. Its carboxy terminal moieties are involved in the recognition and binding of unique cell wall polymers. Mutanolysin lyses Listeria and other gram positive bacteria such as Lactobacillus and Lactococcus); endochitinase A (an enzyme that breaks down glycosidic bonds in chitin.
  • chitinivorous bacteria bacteria that are able to digest chitin
  • chitobiosidase another chitin degrading enzyme
  • N-Acetyl-beta-Glucos-aminidase hyaluronidase
  • hyaluronidase Chodroitin
  • chondroitinase chondroitinase (ABC, AC or C)
  • cysteine proteases have a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad.
  • the first step is deprotonation of a thiol in the enzyme's active site by an adjacent amino acid with a basic side chain, usually a histidine residue.
  • the next step is nucleophilic attack by the deprotonated cysteine's anionic sulfur on the substrate carbonyl carbon.
  • a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed.
  • the thioester bond is subsequently hydrolyzed to generate a carboxylic acid moiety on the remaining substrate fragment, while regenerating the free enzyme.
  • caspases As proteases, they are enzymes that cleave (cut) other proteins.
  • cysteine proteases are called cysteine proteases, because they use a cysteine residue to cut those proteins, and called caspases because the cysteine residue cleaves their substrate proteins at the aspartic acid residue; Germination protease (GPR; an atypical aspartic acid protease located in spore coats); aspartyl protease (a protease which utilizes an aspartic acid residue for catalysis of their peptide substrates. They typically have two highly-conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin. Ex.
  • HIV-1 protease - a major drug-target for treatment of HIV, chymosin (or "rennin”, Renin (with one “n"), cathepsin D, pepsin, plasmepsin, aspartic protease precursor pepsinogen); keratinase and or keratanase.
  • one ore more enzymes used during a step of enzymatic treatment, such as step (a*) can be selected from one or more of the aforementioned enzymes, including any combination.
  • one or more enzymes used during a step of enzymatic treatment is selected from the group of disulfide-bridge altering enzyme, capable of cleaving, linking and/or re-aranging one or more disulfide bridges.
  • Examples of such enzymes that can be suitable according to the invention comprise the following enzyme classes: glutathione-disulfide reductase (EC 1.8.1.7; also known as glutathione reductase, glutathione reductase (NADPH), glutathione S-reductase, GSH reductase, GSSG reductase, NADPH-glutathione reductase, NADPH-GSSG reductase, NADPH:oxidized-glutathione oxidoreductase); protein-disulfide reductase (EC 1.8.1.8, also known as disulfide reductase, insulin-glutathione transhydrogenase, NAD(P)H:protein-disulfide oxidoreductase); thioredoxin-disulfide reductase (EC 1.8.1.9, also known as NADP-thioredoxin reductase, NAD
  • the enzyme in step (a*) is selected from the group consisting of one or more of cell wall-modifying enzyme, cell wall-degrading enzyme, protein-modifying enzyme, protein-degrading enzyme, fat-modifying enzyme, fat-degrading enzyme, disulfide bridge-modifying enzyme, disulfide bridge-oxidizing enzyme, disulfide-bridge-reducing enzyme, and any combination thereof.
  • the enzyme is selected from the group consisting of one or more of cellulase, chitinase, amylase, protease, lipase, reductase and any combination thereof.
  • the enzyme is selected from one or more of any of the enzymes listed in this specification, including any combination thereof.
  • spore or "spores” relates to bacterial spore or spores unless stated otherwise. According to one embodiment of the invention, one or more of fungus, yeast, mould and/or fungal spore is/are successfully sterilized or disinfected.
  • the water-based fluid comprises - in addition to either (i) one or more reductant (s), (ii) one or more enzyme(s), or (iii) a combination of one or more reductant (s) and one or more enzyme(s) - one or more suitable additives, such as salt(s), coenzyme(s), trace element(s), stabilizing agent(s), buffering agent(s), polar, non-polar and zwitterionic detergent(s), surfactant, as well as antimicrobial agent(s) or antimicrobial composition(s).
  • suitable additives such as salt(s), coenzyme(s), trace element(s), stabilizing agent(s), buffering agent(s), polar, non-polar and zwitterionic detergent(s), surfactant, as well as antimicrobial agent(s) or antimicrobial composition(s).
  • one or more enzyme(s) and/or one or more reductant(s) can be provided as a composition for example as powder or tablet, comprising 30% or more phosphate, 5-15% bleaching agents and less than 5% enzyme (percentages are weight percent in relation to the total weight of the composition).
  • one or more enzyme(s) and/or one or more reductant(s) can be provided as a composition, for example as powder or tablet, comprising 15-30% phosphate(s), less than 5% bleaches with oxygen, less than 5% nonionic tensides; less than 5% perfume (for example limonene), and than 5% enzyme (percentages are weight percent in relation to the total weight of the composition).
  • the water-based fluid containing at least one enzyme is provided by dissolving a composition, for example as powder or tablet, comprising 30% or more phosphate, 5-15% bleaching agents and less than 5% enzyme; or 15-30% phosphate(s), less than 5% bleaches with oxygen, less than 5% nonionic tensides; less than 5% perfume (for example limonene),and than 5% enzyme (percentages are weight percent in relation to the total weight of the composition).
  • the water-based fluid comprising one or more enzyme(s) and/or one or more reductant(s) is provided by diluting a concentrated stock solution.
  • the water-based fluid comprising an enzyme is provided by dissolving a composition used for or suitable for household dishwashers.
  • a detergent according to the invention can be any polar- (i.e. ionic, anionic and/or cationic) non-polar-, or zwitterionic detergent or surfactant known in the art, including any combination of one or more polar, non-polar and/or zwitterionic detergent and/or surfactant.
  • a water based fluid comprises SDS (sodium dodecyl sulfate), in a concentration of about 0.5 to 2%, 0.1 to 0.5%, 0.05 to 0.1 %, 0.01 to 0.05%, or less than 0.01 % (weight/volume).
  • Examples of zwitterionic detergents according to the invention comprise, but are not limited to: 3-(N,N-dimethylmyristylammonio)propanesulfonate; 3-(N,N-dimethyloctylammonio)propanesulfonate inner salt; 3-(N,N-dimethyl-palmitylammonio)propanesulfonate; 3-(decyldimethylammonio)propanesulfonate inner salt; 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate; ASB-14; CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate) CHAPSO; EMPIGEN® BB detergent; N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate; and sodium 2,3-dimercaptopropa
  • non-ionic detergents comprise, but are not limited to: 2-cyclohexylethyl ⁇ -D-maltoside; 4-nonylphenoxypolyglycidyl ether; 6-cyclohexylhexyl ⁇ -D-maltoside; Bis(polyethylene glycol bis[imidazoyl carbonyl]); Brij® 30, 35, 56, 72, or 97; chenodeoxycholic acid diacetate methyl ester; Cremophor® EL; cyclohexylmethyl ⁇ -D-maltoside; decaethylene glycol mono-dodecyl ether; decyl ⁇ -D-glucopyranoside; decyl ⁇ -D-maltopyranoside; decyl- ⁇ -D-1-thioglucopyranoside; decyl- ⁇ -D-1-thiomaltopyranoside; diethylene glycol monodecyl ether; diethylene glycol monododecyl ether;
  • anionic detergents comprise, but are not limited to: 1-octanesulfonic acid sodium salt; chenodeoxycholic acid; cholic acid, e.g. from ox or sheep bile; dehydrocholic acid; deoxycholic acid; docusate sodium salt; glycocholic acid hydrate; glycodeoxycholic acid monohydrate; glycolithocholic acid ethyl ester; lithium dodecyl sulphate; lugol solution; N,N-dimethyldodecylamine N-oxide; N-lauroylsarcosine sodium salt; NiaproofTM 4; SODOSIL® RAM 05; SODOSIL® RM 002; SODOSIL® RM 003; SODOSIL® RM 01; SODOSIL® RM 02; sodium 1-butanesulfonate; sodium 1-decanesulfonate; sodium 1-dodecanesulfonate; sodium 1-heptanesulfonate; sodium
  • anionic detergents comprise, but are not limited to: alkyltrimethylammonium bromide; amprolium hydrochloride; benzalkonium chloride; benzethonium chloride; benzethonium hydroxide; benzyldimethylhexadecylammonium chloride; benzyldimethyltetradecyl-ammonium chloride; benzyldodecyldimethylammonium bromide; choline p-toluenesulfonate salt; denatonium benzoate; dimethyldioctadecylammonium bromide; dodecylethyldimethylammonium bromide; dodecyltrimethylammonium bromide; ethylhexadecyldimethylammonium bromide; irard's reagent T; hexadecylpyridinium bromide; hexadecylpyr
  • the water base fluid and/or the processed water comprises one or more additive(s) selected from the group consisting of one or more of salt, acid, base, buffering agent, coenzyme, trace element, stabilizing agent, polar detergent, non-polar detergent, zwitterionic detergent, antimicrobial composition, and any combination thereof.
  • a water based fluid is provided as cold steam and/or water vapour.
  • This can be any water-based fluid of any of the steps (a) and (a*), as well as any rinsing step.
  • One or more enzyme(s) and/or one or more reductant(s) and/or one or more additives can be dissolved or suspended in a water based fluid, which is applied in the form of cold steam vapor and/or as spray.
  • a water-based fluid can comprise e.g. one or more reductant(s) and/or one or more enzyme(s) and/or one or more additives or other compounds can be applied in droplets, which can be comparable (e.g.
  • the invention is not limited to a single, defined composition used for each step, and may be considered flexible and modular in terms of providing several different sterilization or disinfection protocols, according to e.g. the item or group of items to be sterilized, as well as depending on the microbial load, the time span available for sterilizing, tolerances towards various chemicals, temperatures, pressures, sizes and volumes which have to be treated.
  • Sterilization or disinfection according to the invention is a 4 log reduction of microorganisms, spores or microbial burden, more preferably a 5 log reduction and most preferably a 6 log reduction or even more. Sterilization is at least a 6 log reduction, disinfection at least a 4 log reduction, preferably 5 log reduction. Unless stated otherwise, sterilization or disinfection in the context of the present invention relates to surface sterilization or disinfection, respectively.
  • Another important feature of the present invention is the absence of higher temperatures during the sterilization or disinfection process. It can be desirable that the maximum surface temperature on the item to be sterilized or disinfected is below or not exceeding 100°C, 50°C, 37°C, 30°C, 25°C, 20°C, or ambient or room temperature which often is between 20°C and 25°C.
  • the appropriate temperature or temperatures to be selected for the different steps of the invention depend on the efficiency of the different agents used in each step, combined with the temperature sensitivity of the objects or items to be sterilized, the desired level of disinfection (high, medium or low) and the total time available for the combined treatment.
  • each step (a), (a*) and/or (b) is performed using an individual temperature profile per step, said temperature profile being constant or varying or variable during said step.
  • all steps are performed at essentially the same temperature, such as at a temperature not exceeding 50°C, 37°C, 25°C, 20°C, or ambient temperature.
  • the absolute pressure applied is in the range of 1 to 300kPa, 10 to 200kPa, 50 to 150kPa, or 80 to 120kPa during any of the steps (a), (a*), (b) and/or any rinsing step(s).
  • a vacuum or reduced pressure is applied to facilitate providing an essentially water-free environment.
  • the pressure applied corresponds to ambient pressure.
  • the method according to the invention allows for a rapid sterilization.
  • the total time needed for sterilization is not exceeding 60 min, 30 min, 15 min, or 5 min.
  • sterilization or disinfection is achieved in less than 60 min; 30 min; 10 min; or 5 min.
  • These time spans comprise all necessary steps for performing disinfection and/or sterilization from start to finish, thus including one or more step (a), (a*), (b) and any rinsing/washing step(s).
  • step (a) is performed in not more than 20 min, 10 min, 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, or less than 1 min.
  • step (a*) is performed in not more than 20 min; 10 min; 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, or 1 min, or between 20 and 30 min, 15 and 20 min, 10 and 15 min, 12 and 15 min, 10 and 12 min.
  • step (b) is performed in not more than 20 min; 8 min, 7 min, 6 min, 5 min, 4 min, 3 min, 2 min, or 1 min.
  • a rinsing step with processed water or processed gas is performed in not more than 5 min, 4 min, 3 min, 2 min, 1 min, 45 sec, 30 sec, 15 sec, 10 sec, or 5 sec.
  • the total time needed for disinfection and/or sterilization is more than 60 min, such as between 1 hour and 2 hours, or more than 2 hours. In a further embodiment, the total time needed for disinfection and/or sterilization is in the range of 45-60 min, 30-45 min, 20-30min, 10-20 min, 5-10 min or 2-5 min.
  • the total time of treatment with one or more water-based fluid(s), including any washing/rinsing step, i.e. the total time needed for step(s) (a), (a*) and any washing/rinsing step is less than 60 min; 40 min, 30 min; 20 min, 15 min, 10 min; or 5 min.
  • the sterile items may be left in a suitable gas atmosphere, for storage until they are needed.
  • a suitable gas can be selected from the group consisting of one or more of processed gas, ambient air, gas comprising one or more oxidant, ozone, gas comprising one or more reductant, nitrogen, inert as, helium, and any combination thereof.
  • Gases with oxidizing properties which may be used according to the invention may be selected and combined, without limitations from the group consisting of one or more oxygen, ozone, ethylene oxide, hydrogen peroxide, processed gas, ambient air, sterile ambient air, and any combination thereof.
  • ozone is used as gas with oxidizing properties in a substantially water-free environment, such as by the use of an ozone generator known in the art.
  • Ozone can be added to an appropriate gas, such as ambient air.
  • ambient air is sterile, e.g by the use of UV light and/or filtration.
  • ozone concentrations are in the range of 1 to 10000 ppm, 5 to 100 ppm, 10 to 60 ppm, 40-60 ppm, 30-50 ppm, or 15-30 ppm.
  • the ozone concentration is in the range of 1 to 1000 ppm, 10 to 600 ppm, 250 to 450 ppm, or around 350 ppm (i.e. 350 ppm +/-1, +/-2, +/-5 or +/-10 ppm).
  • the ozone concentration applied is dependent on the nature and degree of biological contamination, as well as the susceptibility to oxidative damage of the object to be sterilized.
  • Ozone in gas decays to O 2 with a half-life of approximately 3 days at 20 °C, but as fast as 1.5 seconds at 250 °C. This process may be accelerated by the use of known catalysts.
  • Ozone may also be dissolved in water, where its half-life is considerably shorter, e.g. approximately 30 min at 15 °C or 8 min at or 35 °C at pH 7. Decomposition is faster in a basic environment, e.g. 3 min at pH 10.4 at 15 °C.
  • Ozone may also be converted to oxygen by means of UV light.
  • This process may actually be utilized to sterilize the processed water used for sterilizing/disinfecting according to the invention, as well as to minimize ozone pollution during use of the method or apparatus according to the invention.
  • a second aspect of the invention concerns the use of the method as described above.
  • This method may be used for treatment of one or more items selected from the group comprising laboratory items, medical items, dental items, veterinary items, military items, biological items and/or food processing related items.
  • the method according to the invention may be used for satellites, space rockets and the like, where contamination of space, planets, asteroids, comets has to be avoided, for example in the context of investigating the question of presence or absence of extraterrestrial life forms.
  • An item or object or any part or parts thereof that is/are suitable for sterilization/disinfection according to the invention can be one or more that is/are sensitive to or impaired by one or more of temperature, pH, pressure, radiation and/or oxidation, or any combination thereof, e.g. when subjected to other forms of sterilization/disinfection.
  • such an item or items, or part of said item can be selected from the group consisting of one or more laboratory item(s), medical item(s), dental item(s), military item(s), biological item(s), and food processing related item(s), and any combination thereof.
  • Such items or parts thereof could be selected from the group of medical instruments including instruments used for medical procedures in humans or animals including dental instruments.
  • the item or part of an item to be sterilized/disinfected is an endoscope and/or ultrasound transducer.
  • an item or part of an item is a disposable item.
  • the item is a reusable item.
  • the invention is not limited to reusable items only, and disposable items may be processed as well by the use of the milder and gentler method of sterilization/disinfection according to the invention.
  • disposable items are packaged/wrapped in a container or foil in an ozone containing atmosphere upon disinfection/sterilization, whereby they remain sterile/disinfected until use.
  • Another use of the method according to the invention is application during organ transplantation, such as surface treatment of tissues or organs of human or animal origin prior to implantation.
  • a further use relates to sterilization or disinfection of one or more implants comprising pacemakers, joints, e.g. artificial hips, knees and the like, ligaments, bones, limbs or the like.
  • the method according to the invention is also suitable for decontamination of items, parts of items or surfaces, e.g. in the military context of fighting microbial warfare or after terrorist attack or suspicion of terrorist or military activities comprising microbial activities.
  • a third aspect of the invention relates to an apparatus comprising the necessary means for performing, using or applying the above mentioned method.
  • a single, combined apparatus is used for performing steps of rendering a layer or fraction or subfraction of a layer or coat of the bacterial spore susceptible for the sporicidal/sterilizing action of the second treatment.
  • the invention is not limited to a single apparatus. Different devices may be used for the different steps according to the invention. For example, the rinsing and/or incubations step(s) can be performed in using one specialized apparatus, while the ozone treatment can be carried out in another device, and the time intervals in between the different steps may be selected accordingly.
  • Figure 2 One embodiment of an apparatus according to the invention is shown in Figure 2 .
  • An apparatus can also comprise one or more separate or individual reaction chamber(s) ("container(s)") for sterilizing one or more items ( Fig. 3 ).
  • a container may be removed from said apparatus, and more preferably, the content of such a container will remain sterile upon removal from said apparatus.
  • the whole container, or just a part of it, for example the device (18) in Fig. 3 on which the item to be sterilized (20) rests, may be used in the transportation from one place to another, such as from one apparatus to another apparatus for performing one or more of the following treatments/steps in any order:
  • the apparatus comprises pH-controlling means, pH-adjusting means and/or pH-maintaining means to control, adjust and/or maintain a defined pH in an aqueous fluid according to the invention.
  • said pH-controlling means, pH-adjusting means and/or pH-maintaining means can comprise a pH electrode and one or more titration means for dosing defined amounts of an acid, base or buffering agent, whereby a defined pH or a defined pH interval, e.g. a defined pH +/- 0.01, +/- 0.05, +/- 0.1, +/- 0.5 pH units can be maintained.
  • a pressure higher than ambient pressure is maintained in the container.
  • a container can be equipped with a device or indicator of sterility, in an either manual, semi- or fully-automated fashion.
  • a container can comprise a pressure indicator, indicating that the content of said container is sterilized.
  • FIG. 4 An alternative embodiment of a container according to the invention is illustrated schematically in Fig. 4 .
  • the container for sterilization and/or disinfection consists of a body (14) which is closed with two lids (16, 40). The lumen of the container is held air- and water tight by a seals, and clamping devices .
  • the item or items to be treated (50) rest on a device (52), which is attached to the body (14). By removing both lids, one or several rinsing/washing/incubation steps can be performed, giving access to for washing/rinsing/treatment from top and bottom, if desired.
  • the object/device to be treated while situated on the device (52) surrounded by the body (14), may be placed in a rinsing/washing/incubation device.
  • the lumen (42) of the device can be filled with liquids and gasses via one or more inlets, outlets or in-and outlets (24, 43, 44, 46, 48), which can be situated either on the one or the other lid or both, or on the body (14).
  • FIG. 1 Schematic representation of an apparatus (2) according to the invention with a sterilizing chamber (4), one or more inlets (6) and one or more outlets (8).
  • a lumen (12) is created between the body of the sterilizing chamber (14) and a removable lid (16). Lid (16) and body (14) remain air-tightened by use of a seal (26, 30) and clamping device (28, 32, 34). The object to be sterilized (20) rests on a device (18), which may be detachable from the body (14).
  • Liquids and gases according to the invention are transported into and out of the lumen (12) by one or more inlets and outlets (22, 24).
  • a pressure indicator located on either lid (34) or body of the sterilizing chamber (36) indicates if there is an overpressure present, indicating sterility of the item to be sterilized (20).
  • FIG. 3 Schematic representation of an alternative disinfection/sterilizing chamber according to the invention.
  • a lumen (42) is created between two lids (16 and 40) and the body of the sterilizing chamber (14).
  • the container is air- and water tightened by seals (30) and one or more clamping devices (32, 34).
  • seals (30) and one or more clamping devices 32, 34.
  • the item or object to be sterilized or disinfected (50) rests on a device (52), which is attached to the body (14). By removing both lids, one or several rinsing/washing/incubation steps can be performed, giving access to washing/rinsing/treatment from top and bottom if desired.
  • the lumen (42) of the device can be filled with liquids and/or gasses via one or more inlets, outlets or in-and outlets (24, 43, 44, 46, 48), which can be situated either on the one or the other lid or both, or on the body (14).
  • inlets, outlets or in-and outlets 24, 43, 44, 46, 48
  • a material compatibility test was performed to investigate ozone's corrosivity with respect to materials commonly used for medical equipments, such as ultrasound transducers and flexible endoscopes. Following materials were selected as representatives of different groups of materials: ABS polyurethane TPX, silicone, neoprene rubber and PVC. Materials were tested in a chamber with 40 to 50 ppm (parts per million) ozone at 25°C to 40°C for 80 hours subjected to cycles of one hour in ozone and one hour in air. There were no visible damages or changes to the surface of the material compared to untreated control samples.
  • the sterilization and/or disinfection effect was investigated of a treatment comprising a liquid comprising a reductant and a detergent, followed by a treatment with ozone gas.
  • Bacillus atrophaeus spores are highly resistant to heat and chemicals and their use is officially recognized for sterilization procedure certification. Similarly suture loops and porcelain penicylinders inoculated with Bacillus subtilis spores are used to determine the efficiency of sterilization protocols.
  • Standard growth media were used, such as Nutrient Agar (i.e. Peptone, Meat extract and Agar), or TSA (i.e. Tryptone Soya Agar)
  • the ozone generator provided by the applicant provided ⁇ 350 ppm O 3 , and a flow rate of ⁇ 25 l/min, using setting No. 1.
  • Filtering test In filtering test experiments the spore solutions were prepared in a volume of 1 ml and processed. Thereto, after an incubation period of 5 min in the appropriate buffer at 40°C using a rotary shaker, an aliquot of 250 ⁇ l of the suspension was transferred onto a sterile single use filters. The filter was washed two times with 10 ml of sterile water. The filter was retrieved under sterile conditions, and the filter was cut into halves using a sterile scissor. Only one half of the filter was treated with ozone, while the other half was kept as control, i.e. without ozone treatment. Finally, both halves were placed onto a TSA plate and incubated at 30°C. Initially Nutrient Agar plates were used, but later on a switch was made to TSA plates, in order to utilize the more pronounced reddish colour of Bacillus atrophaeus colonies which was facilitating analysis and documentation.
  • Sterility test In sterility test experiments sutures and penicylinders are individually placed in glass tubes containing 5 ml Nutrient Broth and incubated for 1-2 days at 30°C with shaking. Visual inspection of the tubes is used to monitor growth/no growth.
  • the length of the lag phase i.e. the time period before growth can be observed, is an indicator of the number of viable cells. The longer the lag phase, the lower the number of viable cells, indicating killing, sterilizing or disinfection of the inoculated suture and/or penicylinder.

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0535612A1 (de) * 1991-09-30 1993-04-07 Olympus Optical Co., Ltd. Verfahren und Vorrichtung zum Regenerieren von zur Handhabung von biologischen Substanzen verwendeten Geräten
US20020122753A1 (en) * 1996-06-26 2002-09-05 Ozontech Ltd. Ozone applications for disinfection, purification and deodorization
EP1584923A2 (de) * 2004-04-07 2005-10-12 Roche Diagnostics GmbH Stabilisierung von Biomolekülen in Proben
WO2005112627A1 (en) * 2004-04-20 2005-12-01 Regeneration Technologies, Inc. Process and apparatus for treating implants
WO2005120592A1 (en) * 2004-06-08 2005-12-22 Whiteley Corporation Pty Ltd Biofilm remover
US20080085211A1 (en) * 2006-10-04 2008-04-10 National Cheng Kung University Method for Sterilizing Biological Materials
WO2008055501A1 (en) 2006-11-08 2008-05-15 B-K Medical Aps Method and apparatus for two-step sterilization

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0535612A1 (de) * 1991-09-30 1993-04-07 Olympus Optical Co., Ltd. Verfahren und Vorrichtung zum Regenerieren von zur Handhabung von biologischen Substanzen verwendeten Geräten
US20020122753A1 (en) * 1996-06-26 2002-09-05 Ozontech Ltd. Ozone applications for disinfection, purification and deodorization
EP1584923A2 (de) * 2004-04-07 2005-10-12 Roche Diagnostics GmbH Stabilisierung von Biomolekülen in Proben
WO2005112627A1 (en) * 2004-04-20 2005-12-01 Regeneration Technologies, Inc. Process and apparatus for treating implants
WO2005120592A1 (en) * 2004-06-08 2005-12-22 Whiteley Corporation Pty Ltd Biofilm remover
US20080085211A1 (en) * 2006-10-04 2008-04-10 National Cheng Kung University Method for Sterilizing Biological Materials
WO2008055501A1 (en) 2006-11-08 2008-05-15 B-K Medical Aps Method and apparatus for two-step sterilization

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SETLOW: "Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals", JOURNAL OF APPLIED MICROBIOLOGY, vol. 101, 2006, pages 514 - 525

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